Lipoproteins are the primary carriers of plasma cholesterol, triacylglycerols, and other lipids. The lipoproteins are micellar lipid-protein complexes consisting of a hydrophobic lipid core surrounded by a shell of polar lipids and apoproteins. Lipoproteins are characterized by their buoyant densities which have resulted in identification of at least six major density classes: chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), lipoprotein(a)s (Lp(a)), and high-density lipoproteins (HDL). At least ten apoproteins associated with these lipoproteins have been isolated and characterized: A-1, A-2, A-4, B-48, B-100, C-1, C-2, C-3, E, and apo(a). All of these apoproteins, except B-48 and B-100, are water-soluble and exchange readily between lipoproteins of different classes.
Many studies have now established an inverse relationship between plasma HDL cholesterol levels and risk of coronary artery disease (CAD), i.e., elevated levels of plasma cholesterol found in HDL particles correlate with a reduced risk of CAD. See, e.g., Goldbourt et al., Int. J. Epidemiol., 15:51-55 (1986). Similarly, many studies have shown that plasma levels of apoprotein A-I (apo A-I), the major protein component of HDL, are also inversely related to the risk of CAD.
Because of the inverse correlation of HDL levels with CAD, extensive research has been directed towards determining the role of HDL in lipid metabolism. Functionally, HDL is now believed to mediate the removal of cholesterol released into the plasma from dying cells and from membranes undergoing turnover. It is believed that an acyl transferase in HDL esterifies these cholesterol molecules, which are then rapidly shuttled to VLDL or LDL particles by a transfer protein. See, generally, Stryer L., Biochemistry, 3rd ed., W. H. Freeman and Co., New York, (1988), pp 560-564.
The amino acid residue sequence of human apo A-I has been determined by Edman degradation of the protein [Brewer et al., Biochem. Biophys. Res. Comm., 80:623-630 (1978)] and by sequencing full-length apo A-I cDNA [Seilhamer et al., DNA, 3(4):309 (1984)]. These studies report that mature apo A-I is a single chain protein of 243 amino acid residues.
The conformation of the A-I protein in HDL particles is not known [Curtiss et al., J. Biol. Chem., 263:13779 (1988)]. Additionally, immunochemical characterization of native apo A-I, i.e., apo A-I as it is found on HDL particles, has been problematic because of its antigenic heterogeneity and instability. The antigenic heterogeneity of native apo A-I appears to be due to masking of some epitopes by lipids in the intact HDL particle or due to a dependency of the antibody-binding ability of some epitopes upon conformations of native apo A-I in the presence of lipids or other HDL-associated proteins, e.g., the A-2 protein. The antigenic instability of native apo A-I, as manifested by its changing immunoreactivity over time with defined antisera, appears to be due to such phenomena as self-association and deamidation, both of which have been shown to occur in vitro. See Curtiss et al. in "Proceeding of the Workshop on Lipoprotein Heterogeneity", ed. by Lippel, National Institutes of Health Publication No. 87-2646, pp. 363-377 (1987). Further, the effects of storage and NaOH treatment on native apo A-I immunoreactivity are similar, although not identical, which suggests that other processes may be involved in the loss of immunoreactivity during storage than deamidation alone [Milthorp et al., Arterio., 6:285-296 (1986 )].
The antigenic heterogeneity and instability of native apo A-I have made it difficult to develop immunoassays for native apo A-I in patient vascular fluid samples. This is due, at least in part, to proposed methods requiring the use of a reference material (standard) that has an immunoreactivity with anti-apo A-I antibodies comparable to the immunoreactivity of native apo A-I with the anti-apo A-I antibodies.
Recently, efforts to overcome the problems associated with the antigenic heterogeneity and instability of native apo A-I have focused on using monoclonal antibodies (Mabs) to identify epitopes on native apo A-I molecules, which expression is consistent or "conserved" under specific isolation and storage conditions. Such epitopes are referred to as "conserved native epitopes".
Conserved native apo A-I epitopes are reported to be defined by the 1-15 and 90-105 amino acid sequences of apo A-I [Curtiss et al., J. Biol. Chem., 263:13779 (1988)]. Synthetic polypeptides representing residues 1-15 and 90-105 are reported to inhibit HDL-binding of monoclonal antibodies AI-16 and AI-18, respectively. The synthetic polypeptide representing apo A-I residue sequence 90-105 and the AI-18 monoclonal antibodies are the subjects of U.S. Pat. No. 5,055,396. Other monoclonal antibodies include Mabs AI-4, AI-7, AI-9, and AI-11, which are reported in U.S. Pat. No. 4,677,057 to be immunoreactive with native apo A-I; however, the regions of apo A-I that define the epitopes bound by these antibodies have not been reported. The disclosures of U.S. Pat. No. 5,055,396 and U.S. Pat. No. 4,677,057 are incorporated herein by reference.
Additionally, certain monoclonal antibodies are reported to act as anti-apo A-I "pan" antibodies, i.e., antibodies that bind most, if not all, species of native apo A-I in plasma [Hogle et al., J. Lipid. Res., 29:1221-1229 (1988); Curtiss et al., in "Biotechnology of Dyslipoproteinemias: Clinical Applications in Diagnosis and Control" Lenfant et al., eds, pp. 217-226, Raven Press (New York), 1989]. Exemplary monoclonal antibodies in this regard include AI-10, AI-11, AI-12, AI-13, and AI-14, as discussed in U.S. Pat. No. 5,126,240 which disclosures are incorporated herein by reference. The epitopes on native apo A-I molecules with which these antibodies immunoreact have not been identified.
An antibody composition that immunoreacts with about 90 percent or more of native apo A-2 is referred to herein as a pan antibody composition, or pan antibodies. That composition can contain polyclonal antibodies, but more preferably contains one or more monoclonal antibodies as are described in the before-cited patents and patent applications. The single epitope or plurality of epitopes bound by pan antibodies is referred to herein as a pan epitope.
Contrary to the relationship of HDL levels to CAD, high levels of serum LDL have been correlated with abnormal lipid metabolism and CAD [Brown et al., New Engl. J. of Med., 323(19):1289 (1990). During normal lipid metabolism, LDL particles transport cholesterol to peripheral tissues and regulate de novo cholesterol synthesis at these sites. The particles bind to extracellular LDL receptors at these sites and are taken into the cells by endocytosis [Anderson et al., Cell, 10:351 (1977)]. The particles are chemically broken down within the cell, releasing cholesterol and amino acids. The released cholesterol molecules control feedback mechanisms that regulate de novo cholesterol synthesis and control synthesis of cholesterol receptors by the cells. See, generally, Stryer L., supra.
The basis for the relationship between LDL and CAD is believed to be due primarily to deposition of cholesterol on the arterial intima, in the form of atheromatous plaques, when high concentrations of LDL-cholesterol occur in the plasma. High plasma levels of LDL can occur when the diet is enriched in cholesterol or when certain genetic disorders, such as familial hypercholesterolemia (FH), are present. The molecular defect in most cases of FH is an absence or deficiency of functional LDL receptors. Homozygotes of FH typically have severely elevated cholesterol levels (680 mg/dl) and usually die of CAD in childhood. Heterozygotes (1 in 500 persons) typically show elevated cholesterol levels (300 mg/dl) and have increased CAD risk.
Structurally, LDL particles have a cholesterol core comprising about 1500 esterified cholesterol molecules surrounded by a shell of phospholipids, unesterified cholesterol molecules, and a single molecule of apoprotein B-100 (apo B-100). The hydrophilic shell of an LDL particle facilitates hydration and suspension of the particle in plasma. Cellular LDL receptors recognize and bind to native apo B-100, i.e., as it appears in LDL particles, thereby extracting LDL particles from the plasma. Hence, the B-100 protein is critical for effective receptor-mediated uptake and clearance of LDL particles from circulation. Accordingly, investigators have suggested that plasma levels of native apo B-100 actually may be more predictive of CAD risk than plasma LDL cholesterol levels [Sniderman et al., Proc. Natl. Acad. Sci. U.S.A., 77:604-608 (1980); Sniderman et al., Arteriosclerosis, 10(5):665 (1990); Albers et al., Clinics in Laboratory Medicine, 9(1):137 (1989)].
The B-100 protein is also found in VLDL and IDL particles, which are comprised of endogenous triacylglycerol and cholesterol ester cores, respectively. A fragment of apo B-100, denoted apo B-48, is found in chylomicrons, which transport dietary triacylglycerols and cholesteryl esters. The apo B-48 molecule is reported to correspond to the N-terminal 47 percent (residues 1-2142) of the apo B-100 molecule [Innerarity et al., J. Clin. Invest., 80:1794 (1987); Powell et al., Cell, 50:831 (1987); Chen et al., Eur. J. Biochem., 175:111 (1987)].
The complete amino acid residue sequence (4563 residues; 514 kDa) of human apo B-100, as determined by cDNA clones of hepatic mRNAs, has been reported [Knott et al., Nature 323:734 (1986); Knott et al., Nucl. Acids. Resch., 14:7501 (1986)]. The full cDNA sequence for the human apo B-100 gene, consisting of 29 exons, also has been reported [Ludwig et al., DNA 6:363 (1987)].
As the level of lipoproteins in circulation is related to the level of associated apoprotein, several schemes for assaying the apoprotein have been proposed. Immunoassays for native apo B-100 have been proposed which utilize specific antibody-containing antisera, including competitive fluid phase and solid phase radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and radial immunodiffusion (RID). Problems associated with these immunoassays include reproducibility and the homogeneity and specificity of the antisera used [Currey et al., Clin. Chem., 24:280-286 (1978); Rosseneu et al., Clin. Chem., 28:427-433 (1983); Albers et al. supra].
Several investigators have reported development of monoclonal antibodies against human B-100 apoproteins. The use of anti-apo B-100 monoclonal antibodies for measuring plasma B-100 levels has been proposed [Patton et al., Clin. Chem., 29:1898-1903 (1983); Maynard et al., Clin. Chem., 30:1620-1624 (1984); Young et al., Clin. Chem., 32:1484-1490 (1986); Young et al., Arteriosclerosis, 6:178 (1986)]. In addition, a mixture of anti-apo B-100 monoclonal antibodies in a RID assay for plasma B-100 has been proposed [Marconvina et al., Clin. Chim. Acta, 147:117-125 (1985)]. However, these assay techniques suffer from the requirements of lengthy incubations, repeated centrifugations and/or use of radioactive materials.
A number of antibody-binding domains of apo B-100 have been mapped with anti-apo B-100 antibodies [Knott et al., Nature 323:734 (1986); Krul et al., J.Lipid Res. 29:937 (1988); Pease et al., J.Biol.Chem. 265:553 (1990)]. In these studies, fragments of apo B-100 cDNA were cloned into vectors and expressed as .beta.-galactosidase fusion proteins in E. coli. The fusion proteins were probed with various anti-apo B-100 antibodies. The results of these and other studies suggest that anti-apo B-100 monoclonal antibodies often recognize complex epitopes on native apo B-100 which are not recognized on delipidated apo B-100 [Curtiss et al., J. Biol. Chem., 257:15213 (1982)].
The antigenic heterogeneity of native apo B-100, as with apo A-I, is well documented. For instance, epitope expression on native apo B-100 molecules has been found to be modulated by: (1) the composition of associated lipids; (2) temperature of the immunoreaction; (3) the degree of isolation of LDL from its native environment; and (4) genetic variety among individuals. Thus, the identification of conserved, pan native epitopes of apo B-100 is important to any generally applicable assay method. An antibody composition that immunoreacts with a conserved, pan native apo B-100 epitope is similarly referred to as a pan antibody composition or pan antibodies.
Because of the unique specificity of monoclonal antibodies, their utility in an assay often depends upon the affinity of the antibodies for a target antigen. Moreover, although monoclonal antibodies may have sufficient liquid-phase affinity for an antigen, when the same antibodies are affixed to a surface they may not be as effective in binding the antigen. Hence, while certain monoclonal antibodies have been reported, their utility in a particular assay format must be demonstrated. Similarly, when an antigen such as LDL or other apo B-100-containing material is affixed to a surface the affinity of the immobilized material for antibodies in solution may not be sufficient, even though it is sufficient when the apo B-100-containing material is present in the liquid phase. See, e.g., Goding, J., Monoclonal Antibodies: Principles and Practice, Academic Press, New York (1983), pp 40-46.
Currently proposed methods for assaying the lipoproteins described above are deficient in a number of respects. For instance, immunoassay methods for determining the amount of native apo A-I in a lipoprotein-containing sample are disclosed in U.S. Pat. No. 4,677,057, U.S. Pat. No. 5,126,240 and U.S. Pat. No. 5,055,396. However, these methods do not disclose concurrent determination of other apoprotein levels. On the other hand, U.S. Pat. No. 4,828,986 proposes competitive and "sandwich" immunoassay methods for determining the ratio of apolipoproteins B-100 and A-I using monoclonal antibodies. However, the latter method does not permit assaying for apolipoproteins B-100 and A-I in the same sample aliquot.
Significantly, previously proposed assay methods require the use of "secondary" serum standards in order to make accurate determinations of the assayed apolipoproteins. As used herein, a "secondary standard" refers to: (1) delipidated lipoproteins with added native apoprotein; (2) truncated apoproteins; or (3) freshly isolated pooled plasma collected from normal donors, as described in U.S. Pat. No. 4,828,986. The frequent impracticability of using primary (native) standards is believed to be due to problems associated with their isolation, solubility, stability, and heterogeneity. A review of some of the problems associated with immunoassays of apolipoproteins is presented in Rosseneu et al., Clin. Chem., 24:280-286 (1978); Albers et al., Clinics in Laboratory Medicine, (9)1:137 (1989).
Since the risk of coronary artery disease is known to be correlated to serum levels of HDL and LDL, which levels are related to apo A-I and apo B-100, respectively, an assay method that permits determination of both apoproteins is desired. Preferably, the apoprotein assays could be performed on a single sample aliquot. An assay method that permits ready calibration without the need for secondary serum standards is particularly desired.